Microarrays and particularly nucleic acid microarrays have become important analytical research tools in pharmacological and biochemical research and discovery. Microarrays are miniaturized arrays of points or locations arranged in a column and row format. Molecules, including biomolecules, are attached or synthesized in situ at specific attachment points, which are usually in a column and row format although other formats may be used. An advantage of microarrays is that they provide the ability to conduct hundreds, if not thousands, of experiments in parallel. Such parallelism, as compared to sequential experimentation, can be used to increase the efficiency of exploring relationships between molecular structure and biological function, where slight variations in chemical structure can have profound biochemical effects. Microarrays are available in different formats and have different surface chemistry characteristics that lead to different approaches for attaching or synthesizing molecules. Differences in microarray surface chemistry lead to differences in preparation methods for providing a surface that is receptive to attachment of a presynthesized chemical species or for synthesizing a chemical species in situ. As the name suggests, the attachment points on microarrays are of a micrometer scale, which is generally 1-100 μm.
Research using microarrays has focused mainly on deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) related areas, which includes genomics, cellular gene expression, single nucleotide polymorphisms (SNP), genomic DNA detection and validation, functional genomics, and proteomics (Wilgenbus and Lichter, J. Mol. Med. 77:761, 1999; Ashfari et al., Cancer Res. 59:4759, 1999; Kurian et al., J. Pathol. 187:267, 1999; Hacia, Nature Genetics 21 suppl.:42, 1999; Hacia et al., Mol. Psychiatry 3:483, 1998; and Johnson, Curr. Biol. 26:R171, 1998.) In addition to microarrays for DNA/RNA research, microarrays can be used for research related to peptides (two or more linked natural or synthetic amino acids), small molecules (such as pharmaceutical compounds), oligomers, and polymers.
Considering microarrays for DNA related research, there are numerous methods for preparing a microarray of DNA related molecules. DNA related molecules include native or cloned DNA and synthetic DNA. Synthetic relatively short single-stranded DNA or RNA strands are commonly referred to as oligonucleotides (oligos), which is synonymous with oligodeoxyribonucleotide. Microarray preparation methods include the following: (1) spotting a solution on a prepared flat surface using spotting robots; (2) in situ synthesis by printing reagents via ink jet or other printing technology and using regular phosphoramidite chemistry; (3) in situ parallel synthesis using electrochemically generated acid for deprotection and using regular phosphoramidite chemistry; (4) maskless photo-generated acid (PGA) controlled in situ synthesis and using regular phosphoramidite chemistry; (5) mask-directed in situ parallel synthesis using photo-cleavage of photolabile protecting groups (PLPG); (6) maskless in situ parallel synthesis using PLPG and digital photolithography; and (7) electric field attraction/repulsion for depositing oligos.
Photolithographic techniques for in situ olio synthesis are disclosed in Fodor et al. U.S. Pat. No. 5,445,934 and the additional patents claiming priority thereto. Electric field attraction/repulsion microarrays are disclosed in Hollis et al. U.S. Pat. No. 5,653,939 and Heller et al. U.S. Pat. No. 5,929,208. An electrode microarray for in situ oligo synthesis using electrochemical deblocking is disclosed in Montgomery U.S. Pat. Nos. 6,093,302; 6,280,595, and 6,444,111 (Montgomery I, II, and III respectively), which are incorporated by reference herein. Another and materially different electrode array (not a microarray) for in situ oligo synthesis on surfaces separate and apart from electrodes using electrochemical deblocking is disclosed in Southern U.S. Pat. No. 5,667,667. A review of oligo microarray synthesis is provided by Gao et al., Biopolymers 2004, 73, 579.
Microarrays other than DNA microarrays have been disclosed. For example, the synthetic preparation of a peptide array was originally reported in 1991 using photolithography masking techniques. This method was extended in year 2000 to include an addressable masking technique using photogenerated acids and/or in combination with photosensitizers for deblocking. Reviews of peptide microarray synthesis using photolabile deblocking are provided by Pellois et al., J. Comb. Chem. 2000, 2:355 and Fodor et al., Science, 1991, 251:767. Recent work using peptide arrays has utilized arrays produced by spotting pre-synthesized peptides or isolated proteins. A review of protein arrays is provided by Cahill and Nordhoff, Adv. Biochem. Engin/Biotechnol. 2003, 83:177.
Preparation methods for providing a microarray surface that is receptive to attachment of a presynthesized chemical species or for synthesizing a chemical species in situ must provide a surface that is capable of bonding a chemical species as well as being capable of providing the chemical functionality necessary to conduct pharmacological and biochemical research and discovery. One approach is to treat a surface to provide reactive groups capable of covalently bonding to chemical species of interest. In such an approach, the reactive group is typically present as a result of a surface treatment or coating of the surface. For DNA related species, the reactive group required is a hydroxyl, unless there has been a chemical modification. For peptides, the reactive group required is an amine, unless there has been a chemical modification.
Glass is a commonly used solid substrate for microarrays and must be treated before use. A common glass treatment uses silanization chemistry to introduce a stable and uniform surface having reactive groups for attachment or in situ synthesis of oligos or other chemical species (Guo et al., 1994, Nucl. Acids Res., 22:5456-5465; LeProust et al., 2001, Nucl. Acids Res., 29:2171-2180; Maskos and Southern, 1992, Nucl. Acids Res., 20:1679-1684; Skrzypcznski et al., U.S. Patent Appl. Pub. 2004/0073017, and Southern et al. U.S. Pat. No. 6,576,426.). Glass beads for bulk synthesis must also undergo silanization (Maskos and Southern). Gold surfaces are treated with thiol linker chemistry (Kelley et al. U.S. Patent Pub. 2002/0172963). Similarly, polymeric microarray supports, such as polypropylene, must be treated by oxidation followed by introduction of reactive groups such as terminal amines (Schepinov et al., 1997, Nucl. Acids Res., 25:1155-1161). Additionally, polystyrene beads are surface treated with polyethylene glycols having reactive terminal groups for bulk synthesis of peptides (Merck, Inc. Novabiochem Div. and Aldrich et al., U.S. Patent Appl. Pub. 2003/0134989). Finally, the surface of electrodes on an electrode microarray must be treated with a surface coating to provide reactive groups (Montgomery I, II, and III). For oligo synthesis, such a surface coating must be able to withstand the rigors of repeated exposure to synthesis solutions and to electrochemical deblocking solutions.
The electrochemical synthesis microarray disclosed in Montgomery I, II, and III is based upon a semiconductor chip having a plurality of microelectrodes in a column and row format. This chip design uses Complimentary Metal Oxide Semiconductor (CMOS) technology to create high-density arrays of microelectrodes with parallel addressing for selecting and controlling individual microelectrodes within the array. The electrodes are “turned on” by applying a voltage, which generates electrochemical reagents (particularly acidic protons) that alter the pH in a small, defined “virtual flask” region or volume adjacent to the electrode. In order to provide reactive groups at each electrode, the microarray is coated with a porous matrix material. Biomolecules can be synthesized at any of the electrodes, and such synthesis occurs within the porous matrix material. For the deblocking step, the pH is electrochemically decreased by applying a voltage to an electrode. The pH decreases only in the vicinity of the electrode because the ability of the acidic reagent to travel away from an electrode is limited by natural diffusion and by a buffer in solution.
In general, when a surface is treated, there is a reactive group at the terminal end of a linker that is attached to the surface during treatment. A linker is a molecule that connects a species of interest to a solid surface. For example, a linker for glass has a reactive group at one end and a silane-coupling group at the other for bonding to glass. Linkers can be of various lengths, depending on the particular chemical species used to form the linker. In addition to linkers, spacers can be attached to a linker in order to provide more distance between a solid surface and an attached chemical species. Spacers can be of a different chemistry than linkers. Linkers and spacers for attachment of oligos are disclosed for glass supports (Guo et al., LeProus et. al., Maskos et al., Skrzypcznski and Southern) for aminated polypropylene supports (Schepinov et al.) and for polystyrene beads (Merck, Inc. Novabiochem Div. and Aldrich et al.).
For a surface coating on an electrode microarray, the surface coating itself provides reactive groups that are naturally present within the coating. Montgomery I, II, and III disclose a surface coating comprising controlled porosity glass (CPG); generic polymers, such as, teflons, nylons, polycarbonates, polystyrenes, polyacylates, polycyanoacrylates, polyvinyl alcohols, polyamides, polyimides, polysiloxanes, polysilicones, polynitriles, polyelectrolytes, hydrogels, expoxy polymers, melamines, urethanes and copolymers and mixtures of these and other polymers; biologically derived polymers, polyhyaluric acids, celluloses, and chitons; ceramics, such as, alumina, metal oxides, clays, and zeolites; surfactants; thiols; self-assembled monolayers; porous carbon; and fullerine materials. Montgomery I, II, and III further discloses that the surface coating can be attached to the electrodes by spin coating, dip coating or manual application, or any other acceptable form of coating. Montgomery I, II, and III further discloses linker molecules attached to controlled porosity glass via silicon-carbon bonds and that the linker molecules include aryl, acetylene, ethylene glycol oligomers containing from 2 to 10 monomer units, diamines, diacids, amino acids, and combinations thereof. In each instance, Montgomery discloses coating the entire surface of a microarray device and not just electrode surfaces.
Guo et al. discloses the use of a 23-atom linker for covalently attaching a DNA sequence to glass. The linker is made by reaction of the glass surface with aminopropyltrimethoxysilane to provide an amino-derivatized surface followed by coupling of the amino groups with excess p-phenylenediisothiocyanate to convert the amino groups to amino-reactive phenylisothiocyanate groups. An oligonucleotide is then covalently attached to the amino-reactive group by coupling to the amino-reactive group a 5′ amino-modified oligonucleotide attached to the 5′ end of a sequence of an oligonucleotide. The resulting structure is a solid surface having a linker attached thereto and the linker having an oligonucleotide attached from the 5′ side to the linker. Guo et al. further disclose a spacer comprising up to a 15-deoxythymidylate chain that is between the oligonucleotide and the linker. The spacer has a 5′ amino-modified oligonucleotide to allow attachment to the amino-reactive group. The spacer is attached onto the 5′ end of an oligonucleotide as a part of the oligonucleotide, and then the spacer-oligonucleotide is attached to the linker. As viewed from the glass surface, the final structure provides a glass surface having a linker having attached thereto a 5′ to 3′ prime spacer-oligonucleotide, where the spacer-oligonucleotide has been synthesized elsewhere and then attached to the linker. The 15-deoxythymidylate chain was found to have the highest hybridization signal compared to chains having fewer deoxythymidylate units.
Maskos and Southern disclose silane-coupled linkers for glass. The linkers are different length and are terminated with a hydroxyl for oligonucleotide synthesis on the glass. The linkers are bound to glass through a glycidoxypropyl silane linkage and have a hexaethylene glycol middle section of different lengths. The linkers range from 8 to 26 atoms in length and do not have any charge. Shchepinov et al. discloses spacer molecules for coupling oligonucleotides to aminated polypropylene. The spacer molecules are built using phosphoramidite chemistry and synthesized monomers having diols as a part of the monomeric unit. Both 3′ and 5′ oligonucleotides were built upon the spacers.
LeProust et al. discloses silane linkers terminating in a hydroxyl, amide, or amine group. The linkers were used to synthesize oligonucleotides (deoxythymidylate units) on glass slides to determine the efficiency/fidelity of synthesis. The linkers were nonionic. Southern et al. discloses nonionic linkers/spacers for use on control pore glass (CPG) for oligonucleotide synthesis. The linkers were attached to CPG through a terminal amine attached to a group on the CPG via silanization. Skrzypcznski et al. discloses nonionic linkers/spacer coupled to glass or sol-gel glass coating through silane linkage. The linker/spacer is proposed to have a hydrophobic part next to the glass attached to a hydrophilic part where a DNA probe is attached.
Linkers and spacers are sometimes used for peptide synthesis off of a microarray. Specifically, microscopic polystyrene (PS) beads are used as a solid support (Aldrich et al.). The beads have a polyethylene glycol (PEG) spacer attached to the beads and a linking group attached to the PEG, where the linking group has a reactive group for synthesis of peptides. After synthesis, the peptides are cleaved from the linking group and recovered. Numerous PS-PEG resins for synthesis are available commercially from Merck Company, Novabiochem Division, as well as other sources.
Oligo microarrays made with the electrochemical process as disclosed in Montgomery I, II, and III have had problems with oligo quality, where quality is judged by missing deoxynucleotide bases in sequences resulting from inefficient deblocking. In addition, quality problems can arise from delamination of the coating over the electrodes. Control pore glass coatings and polysaccharide agarose coatings are both prone to delamination quality problems. Such quality problems have caused the resulting oligo microarray to be less useful for sensitivity of gene expression assays (i.e., finding low abundance mRNA species) and for single nucleotide polymorphisms (SNP) assays, wherein single base changes need to be detected. Peptide synthesis on electrode microarrays has also been problematic. Similar quality problems have been found for glass microarrays, where research has found inefficient reactions of the various reagents with functional groups close to glass plate surfaces (LeProust et al.).
Considering (1) the above discussion of electrode microarray quality problems for oligonucleotides, peptides, and other chemical species, and (2) the need for a surface having reactive groups on electrode microarrays, there is a need in the art to be able to improve in situ electrochemical synthesis quality to provide microarrays having higher quality. The present invention addresses these needs. Additionally, for electrode microarrays, there is a need for a modified surface coating incorporating a linker and spacer to improve synthesis quality and prevent fluorescence quenching.